This invention relates to reagents useful as inhibitors of hepatitis B virus (HBV) replication and gene expression.
The following is a discussion of relevant art, none of which is admitted to be prior art to the pending claims. Examples of art discussed below include Kato et al., 266 J. Biol. Chem. 22071, 1991, Price et al., 86 Proc. Natl. Acad. Sci. USA 8541, 1989, and Nassal et al., 63 Cell 1357, 1990.
Although acute hepatitis following perfusion exposure to human serum or blood was recognized more than 100 years ago, a causative agent was not identified until the 1960s when hepatitis B surface antigen (HBsAg) was discovered and subsequently shown to be a viral antigen. Many liver infections are asymptomatic, but the virus is hepatotrophic and may persist for years. Persistent infections are often associated with minimal liver damage, but the risk of cirrhosis or hepatocellular carcinoma in patients carrying HBV can be more than 200 times greater than in non-carriers.
The Hepatitis B virus (HBV) is the prototypic member of the family of viruses known as the hepadnaviridae. The 3.2 kb genome is characterized by extensively overlapping reading frames which express different proteins through the use of internal initiator codons (AUGs). Three major transcripts are synthesized from the HBV genome, but the major pregenomic MRNA (3200 nucleotides in length) is probably the template for DNA synthesis and the translation of the C and P gene products. The transcript from which the X gene is translated is unclear. In principle, any of the three transcripts can function as a polycistronic mRNA and be utilized for the translation of proteins, including the X protein. Although all RNAs contain an 85 nucleotide encapsidation signal near their 5' ends, only the shortest mRNA is encapsidated into core viral particles and used for reverse transcription into viral genomes. Recently, it was demonstrated that the progression of the 80s ribosome through the region containing the encapsidation signal would preclude the functioning of the signal and prevent the encapsidation of an RNA into the core particles. Thus, disruption of this signal and the prevention of ribosomal progression could be utilized to alter protein expression and viral maturation.
Virions of HBV are spherical particles which are surrounded by a lipid envelope and contain the small circular DNA genome of approximately 3200 nucleotides. The virion also contains a DNA polymerase (reverse transcriptase?) activity and the viral core protein (HBcAg). The HBsAg protein is found in the viral envelope and may exist as three different species. The antigenic complexity of the HBsAg is much greater than expected from the three species synthesized. Greater than eight subtypes of HBsAg can be found in clinical isolates. The P gene (polymerase) shares sequence homology with sequences in many retrovirus pol genes. The X gene encodes a polypeptide of unknown function during viral replication, but which can serve as a transactivator of transcription, and thus is a candidate oncoprotein in the morphogenetic transformation of hepatocytes infected with HBV.
Much of the hepatocellular injury which is associated with mild cases of hepatitis is thought to be caused by immunologic defenses, particularly cytotoxic T-cell responses. It has additionally been shown that chronic carriers have impaired ability to induce interferon protection in infected cells. The role of HBV in progression to hepatocellular carcinoma (HCC) is supported epidemiologically, but no gene product has been demonstrated to be essential. In a number of cases the X gene has been found in an integrated form, often with deletions, but the duck HBV contains no X gene and thus the role of X in duck HCC is not supported.
For some disease syndromes there is evidence that HBsAg-anti-HBs complexes play a role in the pathogenesis, and usually these extrahepatic manifestations occur in the form of skin rash, glomerulitis, arthritis, or necrotizing vasculitis. One third of the patients with biopsy proven polyarteritis nodosa have persistent HBV infection. A fraction of the membranous glomerulonephritis cases are associated with chronic acute HBV and persistent HBV infections. Immune complex deposits can be found along the subepithelial surfaces of the glomerular basement membrane. Pathogenesis appears to be tissue damage mediated by immune complexes.
In general, treatment of chronic hepatitis B has been relatively unsuccessful. Corticosteroids and adenine arabinoside have not been beneficial in the treatment of chronic hepatitis B although some patients do respond to treatment with steroids. After cessation of treatment, these patients show a rebound in serum aminotransferase levels due to an immune response to the increased levels of Hepatitis B which accumulate in the blood during steroid treatment.
Interferons have been used to treat chronic Hepatitis B. Alpha-interferon appears to be more effective than beta- or gamma-interferons although its effectiveness as a single drug has yet to be determined.
Although vaccines for HBV exist, attempts to recruit individuals at risk and institute pre-exposure prophylaxis have been unsuccessful. Adequate control of the disease will require vaccination of newborn infants with a subunit or killed virus vaccine. Approval of such a vaccination regimen is distant. Post-exposure prophylaxis using vaccine or vaccine and Hepatitis B immunoglobulin is sufficient to prevent HBV infection following percutaneous inoculation, oral ingestion or direct mucous membrane contact with HBsAG positive material. The timing of the initial dose of immune globulin is crucial to the success of the treatment. There is no precedent for recommending immunoprophylaxis if exposure to HBV has occurred more than 7 days earlier. Thus a need exists for effective treatment of this disease which utilizes antiviral inhibitors which work by mechanisms other than those currently utilized in the treatment of both acute and chronic hepatitis B infections.